Expression of α-gal epitopes on HeLa cells transduced with adenovirus containing α1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.

[1]  L. A. Murphy,et al.  lmmunochemical Studies of the Combining Sites of the Two Isolectins , A 4 and B 4 , Isolated from Bandeiraea simplicifolial , 2003 .

[2]  M. Tanemura,et al.  Synthesis of α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) on human tumor cells by recombinant α1,3galactosyltransferase produced in Pichia pastoris , 2001 .

[3]  Linda,et al.  Frameshift and Nonsense Mutations in a Human Genomic Sequence Homologous to a Murine UDP-Gal:&D-Gal( 1,4)-D-GlcNAc CY( 1,3)-Galactosyltransferase cDNA* , 2001 .

[4]  Green,et al.  The Asparagine-linked Oligosaccharides on Bovine Fetuin , 2001 .

[5]  J. Crandall,et al.  Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat α1,3‐fucosyltransferase IV gene , 2000, Glia.

[6]  C. Smith,et al.  Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions. , 1999, Journal of immunology.

[7]  S. Y. Zhang,et al.  Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase. , 1999, Cancer research.

[8]  C. Smith,et al.  VCAM-1-Dependent Adhesion Under Flow 2 , 6-Linked Sialic Acid Inhibits α Endothelial , 1999 .

[9]  Takeshi Kobayashi,et al.  Inhibitory effect of α(1,2) fucosyltransferase recombinant adenoviral vector on αGal expression , 1998 .

[10]  JM Wilson,et al.  High throughput method for creating and screening recombinant adenoviruses , 1998, Gene Therapy.

[11]  G. Ayala,et al.  Porcine cartilage transplants in the cynomolgus monkey. III. Transplantation of alpha-galactosidase-treated porcine cartilage. , 1998, Transplantation.

[12]  M. Radic,et al.  A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody. , 1998, Transplantation.

[13]  U. Galili,et al.  Natural anti-Gal antibody as a universal augmenter of autologous tumor vaccine immunogenicity. , 1997, Immunology today.

[14]  J. Wilson,et al.  Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy , 1996, Journal of virology.

[15]  U. Galili,et al.  Synthesis of α-Galactosyl Epitopes by Recombinant α1,3Galactosyltransferase for Opsonization of Human Tumor Cell Vaccines by Anti-Galactose , 1996 .

[16]  R. Cummings,et al.  Transcriptional Regulation of 1,3-Galactosyltransferase in Embryonal Carcinoma Cells by Retinoic Acid , 1996, The Journal of Biological Chemistry.

[17]  U. Galili,et al.  Synthesis of alpha-galactosyl epitopes by recombinant alpha1,3galactosyl transferase for opsonization of human tumor cell vaccines by anti-galactose. , 1996, Cancer research.

[18]  U. Galili,et al.  α-Galactosyl (Galα1-3Galβ1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate α1,3galactosyltransferase expressed in E.coli , 1995 .

[19]  U. Galili,et al.  Suppression of α-galactosyl epitopes synthesis and production of the natural anti-Gal antibody: a major evolutionary event in ancestral Old World primates , 1995 .

[20]  E. Gorelik,et al.  Alterations of cell surface carbohydrates and inhibition of metastatic property of murine melanomas by alpha 1,3 galactosyltransferase gene transfection. , 1995, Cancer research.

[21]  J. Platt,et al.  Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection. , 1995, Journal of immunology.

[22]  U. Galili,et al.  Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. , 1994, Glycobiology.

[23]  M. Sandrin,et al.  Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Ari Helenius,et al.  Stepwise dismantling of adenovirus 2 during entry into cells , 1993, Cell.

[25]  U. Galili.  Interaction of the natural anti-Gal antibody with alpha-galactosyl epitopes: a major obstacle for xenotransplantation in humans. , 1993, Immunology today.

[26]  D. Cooper,et al.  Identification of carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. , 1992, Transplantation proceedings.

[27]  N. Shaper,et al.  Murine alpha 1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing. , 1992, The Journal of biological chemistry.

[28]  J. Rothman,et al.  Molecular dissection of the secretory pathway , 1992, Nature.

[29]  U. Galili,et al.  Gene sequences suggest inactivation of alpha-1,3-galactosyltransferase in catarrhines after the divergence of apes from monkeys. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[30]  U. Galili,et al.  The α-galactosyl epitope on mammalian thyroid cells , 1991 .

[31]  E. Jabs,et al.  Characterization of an alpha 1----3-galactosyltransferase homologue on human chromosome 12 that is organized as a processed pseudogene. , 1991, The Journal of biological chemistry.

[32]  R. Cummings,et al.  Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA. , 1990, The Journal of biological chemistry.

[33]  U. Galili,et al.  Distribution of Gal.alpha.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay , 1990 .

[34]  R. Cummings,et al.  Transfer and expression of a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase gene in transfected Chinese hamster ovary cells. Competition reactions between the alpha 1,3-galactosyltransferase and the endogenous alpha 2,3-sialyltransferase. , 1990, The Journal of biological chemistry.

[35]  U. Galili,et al.  Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay. , 1990, Biochemistry.

[36]  R. Cummings,et al.  Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. V. van Tunen,et al.  Bovine alpha 1----3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. , 1989, The Journal of biological chemistry.

[38]  S. Shohet,et al.  Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. , 1988, The Journal of biological chemistry.

[39]  R. Cummings,et al.  Retinoic acid-induced differentiation of the mouse teratocarcinoma cell line F9 is accompanied by an increase in the activity of UDP-galactose: beta-D-galactosyl-alpha 1,3-galactosyltransferase. , 1988, The Journal of biological chemistry.

[40]  S. Shohet,et al.  Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1----3Gal epitope in primates. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[41]  S. Shohet,et al.  The human natural anti-Gal IgG. III. The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies , 1987, The Journal of experimental medicine.

[42]  D. H. van den Eijnden,et al.  Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus. , 1985, The Journal of biological chemistry.

[43]  S. Shohet,et al.  Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1----3)-linked galactose residues , 1985, The Journal of experimental medicine.

[44]  U. Galili,et al.  A Unique Natural Human Igg Antibody with Anti-a-galactosyl Specificity , 2022 .

[45]  A. Betteridge,et al.  Two alpha-3-D-galactosyltransferases in rabbit stomach mucosa with different acceptor substrate specificities. , 1983, European journal of biochemistry.

[46]  I. Goldstein,et al.  An alpha-D-galactosyltransferase activity in Ehrlich ascites tumor cells. Biosynthesis and characterization of a trisaccharide (alpha-D-galactose-(1 goes to 3)-N-acetyllactosamine). , 1981, The Journal of biological chemistry.

[47]  M. Basu,et al.  Enzymatic synthesis of a blood group B-related pentaglycosylceramide by an alpha-galactosyltransferase from rabbit bone marrow. , 1973, The Journal of biological chemistry.